The present invention relates to aromatic organic compounds which are specific, potent and safe inhibitors of the Ca2+-activated potassium channel (Gardos channel) of erythrocytes and/or of mammalian cell proliferation. More particularly, the invention relates to substituted 11-phenyl dibenzazepine compounds capable of inhibiting the Gardos channel of sickle erythrocytes and/or mitogen-induced mammalian cell proliferation. The compounds can be used to reduce sickle erythrocyte dehydration and/or delay the occurrence of erythrocyte sickling or deformation in situ as a therapeutic approach towards the treatment or prevention of sickle cell disease. The compounds can also be used to inhibit mammalian cell proliferation in situ as a therapeutic approach towards the treatment or prevention of diseases characterized by abnormal cell proliferations
Sickle cell disease has been recognized within West Africa for several centuries. Sickle cell anemia and the existence of sickle hemoglobin (Hb S) was the first genetic disease to be understood at the molecular level. It is recognized today as the morphological and clinical result of a glycine to valine substitution at the No. 6 position of the beta globin chain (Ingram, 1956, Nature 178:792-794). The origin of the amino acid change and of the disease state is the consequence of a single nucleotide substitution (Marotta et al., 1977, J. Biol. Chem. 252:5040-5053).
The major source of morbidity and mortality of patients suffering from sickle cell disease is vascular occlusion caused by sickled erythrocytes, which causes repeated episodes of pain in both acute and chronic form and also causes ongoing organ damage with the passage of time. It has long been recognized and accepted that the deformation and distortion of sickle cell erythrocytes upon complete deoxygenation is caused by polymerization and intracellular gelation of sickle hemoglobin, hemoglobin S (Hb S). The phenomenon is well reviewed and discussed by Eaton and Hofrichter, 1987, Blood 70:1245. The intracellular gelation and polymerization of Hb S can occur at any time during erythrocyte""s journey through the vasculature. Thus, erythrocytes in patients with sickle cell disease containing no polymerized hemoglobin S may pass through the microcirculation and return to the lungs without sickling, may sickle in the veins or may sickle in the capillaries.
The probability of each of these events occurring is determined by the delay time for intracellular gelation relative to the appropriate capillary transit time (Eaton et al., 1976, Blood 47:621). In turn, the delay time is dependent upon the oxygenation state of the hemoglobin, with deoxygenation shortening the delay time. Thus, if it is thermodynamically impossible for intracellular gelation to take place, or if the delay time at venous oxygen pressures is longer than about 15 seconds, cell sickling will not occur. Alternatively, if the delay time is between about 1 and 15 seconds, the red cell will likely sickle in the veins. However, if the delay time is less than about 1 second, red cells will sickle within the capillaries.
For red cells that sickle within the capillaries, a number of possible consequent events exist, ranging from no effect on transit time, to transient occlusion of the capillary, to a more permanent blockage that may ultimately result in ischemia or infarction of the surrounding cells and in destruction of the red cell.
It has long been recognized that the cytoplasm of the normal erythrocyte comprises approximately 70% water. Water crosses a normal erythrocyte membrane in milliseconds; however, the loss of cell water causes an exponential increase in cytoplasmic viscosity as the mean cell hemoglobin concentration (MCHC) rises above about 32 g/dl. Since cytoplasmic viscosity is a major determinate of erythrocyte deformability and sickling, the dehydration of the erythrocyte has substantial Theological and pathological consequences. Thus, the physiological mechanisms that maintain the water content of normal erythrocytes and the pathological conditions that cause loss of water from erythrocytes in the blood circulation are critically important. Not surprisingly, regulation of erythrocyte dehydration has been recognized as an important therapeutic approach towards the treatment of sickle cell disease. Since cell water will follow any osmotic change in the intracellular concentration of ions, the maintenance of the red cell""s potassium concentration is of particular importance (Stuart and Ellory, 1988, Brit J. Haematol. 69:1-4).
Many attempts and approaches to therapeutically treating dehydrated sickle cells (and thus decreasing polymerization of hemoglobin S by lowering the osmolality of plasma) have been tried with limited success, including the following approaches: intravenous infusion of distilled water (Gye et al., 1973, Am. J. Med. Sci. 266:267-277); administration of the antidiuretic hormone vasopressin together with a high fluid intake and salt restriction (Rosa et al., 1980, M. Eng. J. Med. 303:1138-1143; Charache and Walker, 1981, Blood 58:892-896); the use of monensin to increase the cation content of the sickle cell (Clark et al., 1982, J. Clin. Invest. 70:1074-1080; Fahim and Pressman, 1981, Life Sciences 29:1959-1966); intravenous administration of cetiedil citrate (Benjamin et al., 1986, Blood 67:1442-1447; Berkowitz and Orringer, 1984, Am. J. Hematol. 17:217-223; Stuart et al., 1987, J. Clin. Pathol. 40:1182-1186); and the use of oxpentifylline (Stuart et al., 1987, J. Clin. Pathol. 40:1182-1186).
Another approach towards therapeutically treating dehydrated sickle cells involves the administration of imidazole, nitroimidazole and triazole antimycotic agents such as Clotrimazole (U.S. Pat. No. 5,273,992 to Brugnara et al.). Clotrimazole, an imidazole-containing antimycotic agent, has been shown to be a specific, potent inhibitor of the Gardos channel of normal and sickle erythrocytes, and to prevent Ca2+-dependent dehydration of sickle cells both in vitro and in vivo (Brugnara et al., 1993, J. Clin. Invest. 92:520-526; De Franceschi et al., 1994, J. Clin. Invest. 93:1670-1676). When combined with a compound which stabilizes the oxyconformation of Hb S, Clotrimazole induces an additive reduction in the clogging rate of a micropore filter and may attenuate the formation of irreversibly sickled cells (Stuart et al., 1994, J. Haematol. 86:820-823). Other compounds that contain a heteroaryl imidazole-like moiety believed to be useful in reducing sickle erythrocyte dehydration via Gardos channel inhibition include miconazole, econazole, butoconazole, oxiconazole and sulconazole. Each of these compounds is a known antimycotic. Other imidazole-containing compounds have been found to be incapable of inhibiting the Gardos channel and preventing loss of potassium.
As can be seen from the above discussion, reducing sickle erythrocyte dehydration via blockade of the Gardos channel is a powerful therapeutic approach towards the treatment and/or prevention of sickle cell disease. Compounds capable of inhibiting the Gardos channel as a means of reducing sickle cell dehydration are highly desirable, and are therefore an object of the present invention.
Cell proliferation is a normal part of mammalian existence, necessary for life itself. However, cell proliferation is not always desirable, and has recently been shown to be the root of many life-threatening diseases such as cancer, certain skin disorders, inflammatory diseases, fibrotic conditions and arteriosclerotic conditions.
Cell proliferation is critically dependent on the regulated movement of ions across various cellular compartments, and is associated with the synthesis of DNA. Binding of specific polypeptide growth factors to specific receptors in growth-arrested cells triggers an array of early ionic signals that are critical in the cascade of mitogenic events eventually leading to DNA synthesis (Rozengurt, 1986, Science 234:161-164). These include: (1) a rapid increase in cystolic Ca2+, mostly due to rapid release of Ca2+ from intracellular stores; (2) capacitative Ca2+ influx in response to opening of ligand-bound and hyperpolarization-sensitive Ca2+ channels in the plasma membrane that contribute further to increased intracellular Ca2+ concentration (Tsien and Tsien, 1990, Annu. Rev. Cell Biol. 6:715-760; Peppelenbosch et al., 1991, J. Biol. Chem. 266:19938-19944); and (3) activation of Ca2+-dependent K+ channels in the plasma membrane with increased K+ conductance and membrane hyperpolarization (Magni et al., 1991, J. Biol. Chem. 261:9321-9327). These mitogen-induced early ionic changes, considered critical events in the signal transduction pathways, are powerful therapeutic targets for inhibition of cell proliferation in normal and malignant cells.
One therapeutic approach towards the treatment of diseases characterized by unwanted or abnormal cell proliferation via alteration of the ionic fluxes associated with early mitogenic signals involves the administration of Clotrimazole. As discussed above, Clotrimazole has been shown to inhibit the Ca2+-activated potassium channel of erythrocytes. In addition, Clotrimazole inhibits voltage- and ligand-stimulated Ca2+ influx mechanisms in nucleated cells (Villalobos et al., 1992, FASEB J. 6:2742-2747; Montero et al., 1991, Biochem. J. 277:73-79) and inhibits cell proliferation both in vitro and in vivo (Benzaquen et al., 1995, Nature Medicine 1:534-540). Recently, Clotrimazole and other imidazole-containing antimycotic agents capable of inhibiting Ca2+-activated potassium channels have been shown to be useful in the treatment of arteriosclerosis (U.S. Pat. No. 5,358,959 to Halperin et al.), as well as other disorders characterized by unwanted or abnormal cell proliferation.
As can be seen from the above discussion, inhibiting mammalian cell proliferation via alteration of ionic fluxes associated with early mitogenic signals is a powerful therapeutic approach towards the treatment and/or prevention of diseases characterized by unwanted or abnormal cell proliferation. Compounds capable of inhibiting mammalian cell proliferation are highly desirable, and are therefore also an object of the present invention.
These and other objects are provided by the present invention, which in one aspect provides a novel class of organic compounds which are potent, selective and safe inhibitors of the Ca2+-activated potassium channel (Gardos channel) of erythrocytes, particularly sickle erythrocytes, and/or of mammalian cell proliferation. The compounds are generally substituted 11-phenyl-dibenzazepine compounds. In one illustrative embodiment, the compounds capable of inhibiting the Gardos channel and/or mammalian cell proliferation according to the invention are compounds having the structural formula: 
or pharmaceutically acceptable salts of hydrates thereof, wherein:
R1 is xe2x80x94Rxe2x80x2, (C6-C20) aryl or substituted (C6-C20) aryl;
R2 is xe2x80x94Rxe2x80x2, xe2x80x94ORxe2x80x2, xe2x80x94SRxe2x80x2, halogen or trihalomethyl;
R3 is xe2x80x94Rxe2x80x2, xe2x80x94ORxe2x80x2, xe2x80x94SRxe2x80x2, halogen or trihalomethyl or, when taken together with R4, is (C6-C20) aryleno;
R4 is xe2x80x94Rxe2x80x2, xe2x80x94ORxe2x80x2, xe2x80x94SRxe2x80x2, halogen or trihalomethyl or, when taken together with R3, is (C6-C20) aryleno;
each of R5, R6, R7, R9, R10, R11, R12, R13 and R14 is independently selected from the group consisting of xe2x80x94Rxe2x80x2, halogen and trihalomethyl;
R15 is xe2x80x94Rxe2x80x3, xe2x80x94C(O)Rxe2x80x3, xe2x80x94C(S)Rxe2x80x3, xe2x80x94C(O)ORxe2x80x3, xe2x80x94C(S)ORxe2x80x3, xe2x80x94C(O)SRxe2x80x3, xe2x80x94C(S)SRxe2x80x3, xe2x80x94C(O)N(Rxe2x80x3)2, xe2x80x94C(S)N(Rxe2x80x3)2, xe2x80x94C(O)C(O)Rxe2x80x3, xe2x80x94C(S)C(O)Rxe2x80x3, xe2x80x94C(O)C(S)Rxe2x80x3, xe2x80x94C(S)C(S)Rxe2x80x3, xe2x80x94C(O)C(O)ORxe2x80x3, xe2x80x94C(S)C(O)ORxe2x80x3, xe2x80x94C(O)C(S)ORxe2x80x3, xe2x80x94C(O)C(O)SRxe2x80x3, xe2x80x94C(S)C(S)ORxe2x80x3, xe2x80x94C(S)C(O)SRxe2x80x3, xe2x80x94C(O)C(S)SRxe2x80x3, xe2x80x94C(S)C(S)SRxe2x80x3, xe2x80x94C(O)C(O)N(Rxe2x80x3)2, xe2x80x94C(S)C(O)N(Rxe2x80x3)2, xe2x80x94C(O)C(S)N(Rxe2x80x3)2 or xe2x80x94C(S)C(S)N(Rxe2x80x3)2;
each Rxe2x80x2 is independently selected from the group consisting of xe2x80x94H, (C1-C6) alkyl, (C1-C6) alkenyl and (C1-C6) alkynyl;
each Rxe2x80x3 is independently selected from the group consisting of xe2x80x94H, (C1-C6) alkyl, (C1-C6) alkenyl, (C1-C6) alkynyl, (C6-C20) aryl, substituted (C6-C20) aryl, (C6-C26) alkaryl and substituted (C6-C26) alkaryl; and
the aryl and alkaryl substituents are each independently selected from the group consisting of xe2x80x94CN, xe2x80x94ORxe2x80x2, xe2x80x94SRxe2x80x2, xe2x80x94NO2, xe2x80x94NRxe2x80x2Rxe2x80x2, halogen, (C1-C6) alkyl, (C1-C6) alkenyl, (C1-C6) alkynyl and trihalomethyl.
In another aspect, the present invention provides pharmaceutical compositions comprising one or more compounds according to the invention in admixture with a pharmaceutically acceptable carrier, excipient or diluent. Such a preparation can be administered in the methods of the invention.
In still another aspect, the invention provides a method for reducing sickle erythrocyte dehydration and/or delaying the occurrence of erythrocyte sickling or deformation in situ. The method involves contacting a sickle erythrocyte in situ with an amount of at least one compound according to the invention, or a pharmaceutical composition thereof, effective to reduce sickle erythrocyte dehydration and/or delay the occurrence of erythrocyte sickling or deformation. In a preferred embodiment, the sickle cell dehydration is reduced and erythrocyte deformation is delayed in a sickle erythrocyte that is within the microcirculation vasculature of a subject, thereby preventing or reducing the vaso-occlusion and consequent adverse effects that are commonly caused by sickled cells.
In still another aspect, the invention provides a method for the treatment and/or prevention of sickle cell disease in a subject, such as a human. The method involves administering a prophylactically or therapeutically effective amount of at least one compound according to the invention, or a pharmaceutical composition thereof, to a patient suffering from sickle cell disease. The patient may be suffering from either acute sickle crisis or chronic sickle cell episodes.
In yet another aspect, the invention provides a method for inhibiting mammalian cell proliferation in situ. The method involves contacting a mammalian cell in situ with an amount of at least one compound according to the invention, or a pharmaceutical composition thereof, effective to inhibit cell proliferation. The compound or composition may act either cytostatically, cytotoxically or a by a combination of both mechanisms to inhibit proliferation. Mammalian cells that can be treated in this manner include vascular smooth muscle cells, fibroblasts, endothelial cells, various types of pre-cancer cells and various types of cancer cells.
In still another aspect, the invention provides a method for treating and/or preventing unwanted or abnormal cell proliferation in a subject, such as a human. In the method, at least one compound according to the invention, or a pharmaceutical composition thereof, is administered to a subject in need of such treatment in an amount effective to inhibit the unwanted or abnormal mammalian cell proliferation. The compound and/or composition may be applied locally to the proliferating cells, or may be administered to the subject systemically. Preferably, the compound and/or composition is administered to a subject that has a disorder characterized by unwanted or abnormal cell proliferation. Such disorders include, but are not limited to, cancer, epithelial precancerous lesions, non-cancerous angiogenic conditions or arteriosclerosis.
In a final aspect, the invention provides a method for the treatment and/or prevention of diseases that are characterized by unwanted and/or abnormal mammalian cell proliferation. The method involves administering a prophylactically or therapeutically effective amount of at least one compound according to the invention, or a pharmaceutical composition thereof, to a subject in need of such treatment. Diseases that are characterized by abnormal mammalian cell proliferation which can be treated or prevented by way of the methods of the invention include, but are not limited to, cancer, blood vessel proliferative disorders, fibrotic disorders and arteriosclerotic conditions.
3.1 Definitions
As used herein, the following terms shall have the following meanings:
xe2x80x9cAlkyl:xe2x80x9d refers to a saturated branched, straight chain or cyclic hydrocarbon radical. Typical alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl, isopentyl, hexyl, and the like. In preferred embodiments, the alkyl groups are (C1-C6) alkyl, with (C1-C3) being particularly preferred.
xe2x80x9cSubstituted Alkyl:xe2x80x9d refers to an alkyl radical wherein one or more hydrogen atoms are each independently replaced with other substituents. Typical substituents include, but are not limited to, xe2x80x94OR, xe2x80x94SR, xe2x80x94NRR, xe2x80x94CN, xe2x80x94NO2, -halogen and -trihalomethyl, where each R is independently xe2x80x94H, alkyl, alkenyl, alkynyl, aryl or alkaryl as defined herein.
xe2x80x9cAlkenyl:xe2x80x9d refers to an unsaturated branched, straight chain or cyclic hydrocarbon radical having at least one carbon-carbon double bond. The radical may be in either the cis or trans conformation about the double bond(s). Typical alkenyl groups include, but are not limited to, ethenyl, propenyl, isopropenyl, butenyl, isobutenyl, tert-butenyl, pentenyl, hexenyl and the like. In preferred embodiments, the alkenyl group is (C1-C6) alkenyl, with (C1-C3) being particularly preferred.
xe2x80x9cSubstituted Alkenyl:xe2x80x9d refers to an alkenyl radical wherein one or more hydrogen atoms are each independently replaced with other substituents. Typical substituents include, but are not limited to, xe2x80x94OR, xe2x80x94SR, xe2x80x94NRR, xe2x80x94CN, xe2x80x94NO2, -halogen and -trihalomethyl, where each R is independently xe2x80x94H, alkyl, alkenyl, alkynyl, aryl or alkaryl as defined herein.
xe2x80x9cAlkynyl:xe2x80x9d refers to an unsaturated branched, straight chain or cyclic hydrocarbon radical having at least one carbon-carbon triple bond. Typical alkynyl groups include, but are not limited to, ethynyl, propynyl, butynyl, isobutynyl, pentynyl, hexynyl and the like. In preferred embodiments, the alkynyl group is (C1-C6) alkynyl, with (C1-C3) being particularly preferred.
xe2x80x9cSubstituted Alkynyl:xe2x80x9d refers to an alkynyl radical wherein one or more hydrogen atoms are each independently replaced with other substituents. Typical substituents include, but are not limited to, xe2x80x94OR, xe2x80x94SR, xe2x80x94NRR, xe2x80x94CN, xe2x80x94NO2, -halogen and -trihalomethyl, where each R is independently xe2x80x94H, alkyl, alkenyl, alkynyl, aryl or alkaryl as defined herein.
xe2x80x9cAryl:xe2x80x9d refers to an unsaturated cyclic hydrocarbon radical having a conjugated xcfx80 electron system. Typical aryl groups include, but are not limited to, penta-2,4-diene, phenyl, naphthyl, anthracyl, azulenyl, indacenyl, and the like. In preferred embodiments, the aryl group is (C1-C20) aryl, with (C5-C10) being particularly preferred.
xe2x80x9cSubstituted Arvl:xe2x80x9d refers to an aryl radical wherein one or more hydrogen atoms are each independently replaced with other substituents. Typical substituents include, but are not limited to, xe2x80x94OR, xe2x80x94SR, xe2x80x94NRR, xe2x80x94CN, xe2x80x94NO2, -halogen and -trihalomethyl where each R is independently xe2x80x94H, alkyl, alkenyl, alkynyl, aryl or alkaryl as defined herein.
xe2x80x9cAryleno:xe2x80x9d refers to an aryl radical that is capable of fusing to another aryl group. Typical aryleno groups include, but are not limited to, benzeno, naphthaleno, anthracaleno and the like. In preferred embodiments, the aryleno group is (C6-C20) aryleno.
xe2x80x9cSubstituted Aryleno:xe2x80x9d refers to an aryleno group wherein one or more hydrogen atoms are each independently replaced with other substituents. Typical substituents include, but are not limited to, xe2x80x94OR, xe2x80x94SR, xe2x80x94NRR, xe2x80x94CN, xe2x80x94NO2, -halogen and -trihalomethyl, where each R is independently xe2x80x94H, alkyl, alkenyl, alkynyl, aryl or alkaryl as defined herein.
xe2x80x9cAlkaryl:xe2x80x9d refers to a straight-chain alkyl, alkenyl or alkynyl group wherein one of the hydrogen atoms bonded to a terminal carbon is replaced with an aryl moiety. Typical alkaryl groups include, but are not limited to, benzyl, benzylidene, benzylidyne, benzenobenzyl, naphthalenobenzyl and the like. In preferred embodiments, the alkaryl group is (C6-C26) alkaryl, i.e., the alkyl, alkenyl or alkynyl moiety of the alkaryl group is (C1-C6) and the aryl moiety is (C5-C20). In particularly preferred embodiments the alkaryl group is (C6-C13), i.e., the alkyl, alkenyl or alkynyl moiety of the alkaryl group is (C1-C3) and the aryl moiety is (C5-C10).
xe2x80x9cSubstituted Alkaryl:xe2x80x9d refers to an alkaryl radical wherein one or more hydrogen atoms on the aryl moiety of the alkaryl group are each independently replaced with other substituents. Typical substituents include, but are not limited to, xe2x80x94OR, xe2x80x94SR, xe2x80x94NRR, xe2x80x94CN, xe2x80x94NO2, -halogen and -trihalomethyl, where each R is independently xe2x80x94H, alkyl, alkenyl, alkynyl, aryl or alkaryl as defined herein.
xe2x80x9cIn Situ:xe2x80x9d refers to and includes the terms xe2x80x9cin vivo,xe2x80x9d xe2x80x9cex vivo,xe2x80x9d and xe2x80x9cin vitroxe2x80x9d as these terms are commonly recognized and understood by persons ordinarily skilled in the art. Moreover, the phrase xe2x80x9cin situxe2x80x9d is employed herein in its broadest connotative and denotative contexts to identify an entity, cell or tissue as found or in place, without regard to its source or origin, its condition or status or its duration or longevity at that location or position.